This R35 application proposes a conceptual framework in which a body of work produced by the PI over the past 20 years is utilized as the foundation for launching innovative studies that seek to incorporate cutting edge understanding of nuclear architecture (how chromatin is organized in three dimensions in the nucleus) to produce a novel paradigm of lineage determination and cell fate identity. The goal is to better understand how broad gene expression programs that characterize cell identity are regulated in order to inform regenerative approaches to cardiovascular disease. Preliminary data suggest that regions of the genome that are localized to the nuclear periphery (called ?lamin associated domains? or LADs) are silenced by specific histone marks, including H3K9me2, and that these regions are released from the periphery upon lineage determination in order to allow for simultaneous activation of entire gene programs. Data suggests that the H3K9me2 mark characteristic of LADs is ?remembered? through mitosis providing a mechanism for epigenetic memory of lineage identity. The proposed model suggests that epigenetic marks such as H3K9me2 that define LADs are recognized by ?LAD-tethers? that mediate spatial localization, and that these histone epitopes can be ?shielded? by phosphorylation of adjacent amino acid residues of the histone tails (including phosphorylation of H3S10 and H3T11). We propose to test that during mitosis, aurora B kinase which phosphorylates H3S10, acts to un-tether LADs by shielding the H3K9me2 epitope, allowing for the release of LADs, subsequent breakdown of the nuclear membrane and DNA replication, followed by removal of S10 phosphorylation and re-establishment of LADs as the daughter nuclear membranes form around the exposed histone mark. Thus, if the genome-wide pattern of LADs in a given cell defines its identity by representing a ?code? of silenced alternative lineage programs, then cellular identity can be remembered through mitosis and efficiently re-established in daughter cells. Implications for reprogramming, trans-differentiation, asymmetric cell division, and stability of lineage identity (and thus cancer susceptibility) will be explored. Signal transduction cascades that regulate dramatic changes in cellular metabolism and function (such as the switch between glucose and fatty acid metabolism characteristic of developing and ailing cardiac myocytes and of cancer cells) may impact nuclear architecture and LAD dynamics by converging on phosphorylation of histone residues including H3T11. This notion is supported by published data indicating that a nuclear form of pyruvate kinase that is implicated in metabolic shifts can phosphorylate H3T11 and can interact with Hdac3 which we have shown is a LAD tether, resulting in epigenetic changes and activation of specific gene loci. Thus, this proposal provides the opportunity to provide experimental support for a model of gene regulation and cellular identity that incorporates three dimensional regulation of chromatin packaging within the nucleus extending our understanding of cellular identity and providing a novel mechanism to understand the way in which entire gene programs are coordinately regulated.